Digital PCR is a nucleic acid amplification and detection method that is based on the dilution of template DNA into independent non-interacting partitions (Sykes, et al. (1992) BioTechniques 13: 444-449). Following Poisson statistics with high dilutions of DNA template, each reaction is independently interrogated for the presence of a nucleic acid at single molecule sensitivity. Digital PCR was first implemented on high dilutions of template DNA into microtiter plates, but has recently matured through the use of microfabricated platforms (Vogelstein et al. (1999) Proc. Natl. Acad. Sci. U.S.A. 96(16):9236-9241; Ottesen et al. (2006) Science 314(5804):1464-1467; Warren et al. (2006) Proc. Natl. Acad. Sci. U.S.A. 103(47):17807-17812; Heyries et al. (2011) Nat. Methods. 8(8):649-651; Kiss et al. (2008) Anal. Chem. 80(23):8975-8981; Beer et al. (2008) Anal. Chem. 80(6):1854-1858; Shen et al. (2010) Lab Chip 10(20):2666-2672; and Hindson et al. (2011) Anal. Chem. 83(22):8604-8610). In recent years several companies have produced commercially accessible ways to automate and expand the range of partitioning. This included droplet digital PCR (ddPCR) systems (e.g. Bio-Rad QX200) that disperse template DNA randomly into emulsion droplets of equal volume (Hindson et al., supra).
Recently, digital PCR has seen wider use as an analytical tool for research and clinical applications. For example, digital PCR can be used as a robust tool for analyzing copy number variations seen in the amplifications or deletions of specific genes, detecting mutations, and quantifying specific nucleic acids species. Digital PCR has proven useful for identifying cancer genetic variation from tumors; frequently, these samples are admixtures between normal and tumor DNA.
Commonly, ddPCR platforms rely upon the use of fluorescently quenched oligonucleotide probes to hybridize to a region of interest. Upon PCR amplification, the 5′ exonuclease activity of the polymerase separates the fluorophore from the quencher and generates a fluorescent signal specific to the target. The fluorescence of these partitions can be individually measured after amplification in order to determine the presence or absence of template molecules. The use of different fluorescent dyes allows for the simultaneous normalization of one genomic DNA region of interest (ROI) against a reference amplicon in a single reaction. However, the major limitation of using fluorescent oligonucleotide probes in digital copy number analysis is the scalability of synthesis and optimization for a large number of genes.
Recent studies have explored the application of DNA binding dyes such as EvaGreen (EG) for the quantitation of single amplicons in a digital PCR format (Shen et al., supra; Shen et al. (2011) J. Am. Chem. Soc. 133(44):17705-17712). The EG fluorophore is a non-specific double-stranded DNA (dsDNA) binding dye. When no DNA is present, EG assumes an inactive configuration, emitting a fluorescent signal only when template is bound. The method of binding allows for the use of a higher concentration without inhibiting PCR and thus maintaining a higher resolution signal compared to SYBR dye (Eischeid (2011) BMC Res. Notes 4:263). The EG-DNA complex produces a maximum amplitude fluorescent signal at an excitation wavelength of 500 nm and an emission wavelength of 530 nm (Mao et al. (2007) BMC Biotechnol. 7:76). In comparison, digital PCR systems utilizing fluorescent oligonucleotide probes commonly consist of multiple spectrally distinct fluorophores for the detection of different targets. As only one wavelength can be used in an EG-based digital PCR format to detect both the reference and ROI, new multiplexing strategies independent of spectral context must be developed.
Thus, there remains a need for the development of efficient, effective strategies for performing digital PCR analysis.